1Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

Abstract

Cadherins are multidomain adhesion proteins whose interactions direct cell sorting during histogenesis. They determine cell adhesion specificity, but prior studies failed to identify physical differences that could underlie cell sorting. These single molecule studies identify kinetic and strength differences between different cadherins. They further demonstrate that the modular extracellular architecture of cleavage stage C-cadherin supports a multistate binding mechanism. These multiple bonds exhibit a kinetic hierarchy of strengths that map to the different cadherin domains. The outer two N-terminal domains of C-cadherin form two bound states with dissociation rates of 3.9 and 0.02 s(-1). The latter is 25-fold slower than between the corresponding epithelial cadherin segments. In addition to the two fast bonds, the five-domain fragment (CEC1-5) forms two additional stronger, longer-lived bonds with dissociation rates of 0.00039 and 0.00001 s(-1). We further quantified the lifetimes of bonds subject to a constant force, and thus identified multiple dissociation events with rates that agree quantitatively with the force spectroscopy data. The qualitative features are similar to those reported for epithelial cadherin. However, the significant differences in the dissociation rates of the outer domains, which include the specificity-determining region, suggest that kinetic differences may determine cadherin discrimination, rather than adhesion energies.

(A) Structure of the extracellular domain of C-cadherin. (B) Proposed trans adhesive interface observed in the crystal structure of the full C-cadherin ectodomain (8). The intermolecular bond involves EC1 domains from both molecules. The molecular representation was generated with VMD (36).

(A) Biomembrane force probe. The upper panel shows the aspirated RBC with the attached bead and the probe bead held by the second micropipette. The probe bead is brought in and out of contact with the bead on the RBC. The lower panel is a cartoon of the bound cadherins at the bead-bead junction. (B–D) Representative force versus time profiles obtained with the steady-ramp (B), jump/ramp (C), and constant force (D) modes of the BFP.

Measured strengths of trans-bonded CEC12 fragments. (A) Histogram obtained from a steady ramp test of CEC12 versus CEC12 at 69 ± 5 pN/s. The solid lines correspond to the probability distribution at 69 ± 5 pN/s. See text and Table 1 for corresponding bond parameters. (B) Most probable rupture force of the prominent peak (green) versus the logarithm of the loading rate. (C and D) Force histograms obtained with the jump/ramp mode of the BFP. (C) Shows the bonds that failed during the jump phase along with the probability distribution that best fit the data (orange curve). The majority of attachments (∼60%) survived the jump and failed during the ramp phase. (D) Shows the corresponding histogram of rupture forces measured in the ramp phase. The solid green curve is the probability distribution defined by the corresponding bond parameters for this bound state.

Mechanical strengths of CEC1-5 bonds. (A) Histogram of CEC1-5 versus CEC1-5 rupture forces measured under a steady ramp of 68 ± 5 pN/s. The solid lines are the probability distributions for the small fraction (∼30%) of rupture events attributed to the bound states measured between CEC12 fragments: namely, the orange line corresponds to the bound state with Fβ = 5.2 and koff = 3.9 s−1, and the green line corresponds to the bond with Fβ = 5.3 and koff = 0.02 s−1. The blue and red solid curves correspond to the stronger bound states exhibited by CEC1-5. (B) The most probable rupture force corresponding to the prominent peak in the CEC1-5 force histograms versus the logarithm of the loading rates. (C) Force histogram measured during the jump phase (4103 ± 591 pN/s) in the jump/ramp measurements. The majority of bonds (∼60%) survived the jump and failed during the ramp phase (7.8 ± 0.9 pN/s) shown in D. The corresponding histogram shows two, resolved peaks. The peak described by the blue curve is the same as the peak exhibiting the linear dependence shown in A. The second peak was fit by the probability distribution shown by the red curve. The bond parameters pertaining to the red and blue distributions are given in the text and summarized in Table 1.

The survival probability versus the rupture time for CEC1-5 bonds held at 40 pN. The circles are the experimentally measured survival probabilities. The solid line through the data is the superposition of the three exponential functions indicated by the dashed (A1), dotted (A2), and dash-dot curves (A1).

(A) Logarithm10 of the survival probability Psurv versus rupture time for the CEC1-5 bonds held at 40 pN. The lines indicate the three exponential decay curves shown in Fig. 5. (B) Map between the exponential decay functions and the probability distributions describing the peaks in the CEC1-5 force histogram.

Proposed model for cadherin junction assembly. (A) Cadherins rapidly bind via their outer domains and kinetically discriminate between different cadherins (shaded and open). Differences in the binding kinetics of different classical cadherins may provide the kinetic proofreading that underlies selective cadherin binding. (B) Once the proteins engage, the cell membranes are held in close proximity sufficiently long for the slower, strong bonds to form between overlapping proteins. In this staged cadherin junction assembly, the sequence of binding events is governed by the hierarchy of kinetic rates.